1. FIELD OF THE INVENTION
This invention relates to the molecular cloning of the gene encoding Mycoplasma pneumonias P1 cytadhesin protein. This protein mediates mycoplasmal colonization of host respiratory epithelium and is a critical virulence determinant. By the present invention, a complete DNA sequence of the complete P1 gene as well as a deduced amino acid sequence of the P1 cytadhesin protein is presented for the first time. In addition, clones expressing M. pneumonias peptides are provided. Those peptides contain the functional cytadhesin epitopes and have been used to localize the cytadhesin binding domain of P1.
2. DESCRIPTION OF THE RELATED ART
M. pneumonias is a non-invasive pathogen that colonizes the mucosal surface of the respiratory tract and causes a primary, atypical pneumonia. Although this disease appears to occur most frequently in young adults and children, its incidence in the general population may be underestimated because the symptoms are often relatively mild and diagnostic procedures are suboptimal.
M. pneumonias initiates infection by colonizing cells of the respiratory epithelium. This colonization is mediated by a specialized tip-like organnelle containing clusters of a surface-localized, trypsin sensitive protein designated P1. Numerous studies show P1 to be a critical virulence determinant. For example, mutants of M. pneumonlae that lack P1 or are unable to mobilize and anchor P1 at the tip are avirulent. In addition, treatment of virulent M. pneumonias with trypsin abrogates adherence to the respiratory epithelium. Finally, monoclonal antibodies to P1 have been shown to block M. pneumoniae cytadherence. Plummer, et al., Infect. Immun., 53:398-403 (1986).
Unfortunately, despite the critical importance of P1 as a mycoplasmal virulence determinant, efforts to provide a cloned gene encoding the P1 cytadhesin have been generally unsatisfactory. For example, Trevino, et al., Infect. Immun., 53:129-134 (1986), describe an attempt to clone M. pneumonias antigens by constructing an M. pneumonias genomic library using lambda phage EMBL3 as the vector and immunoscreening the library with adsorbed anti-M. pneumonias serum. Although this procedure produced several clones exhibiting antigenic cross-reactivity with M. pneumonias P1, none of the clones reacted with monoclonal antibodies specific for critical antigenic determinants of P1 shown by the present inventors to mediate cytadherence. Moreover, the largest immunoreactive protein identified had a molecular weight of only 140 kDa. In contrast, native P1 has a molecular weight of approximately 165 kDa. Therefore, it could not be definitely established whether or not the 140 kDa protein was a product of the structural P1 gene. The approach was then abandoned.
Since the P1 cytadhesin is probably the most important mediator of mycoplasma cytadsorption, further elucidation of the structure of this molecule is likely to provide information essential for a complete understanding of the role of cytadherence in pathogenesis of mycoplasmal disease. This goal can be achieved most readily by cloning and sequencing the structural gene encoding P1. Furthermore, recent studies have shown that adherence of mycoplasma to respiratory epithelium can be inhibited by certain antibodies directed against cytadhesin epitopes of P1. Therefore, vaccines comprising recombinant P1 protein or selected cytadhesin polypeptides derived from recombinant P1 are likely to prove effective in preventing mycoplasmal infection. In addition, the availability of the complete gene sequence and deduced amino acid sequence for M. pneumonias P1 will allow one to map critical antigenic epitopes and produce selected synthetic peptides useful as diagnostic probes or vaccines.